An oncolytic virus is a type of conditionally replication-competent viruses, which selectively replicates in tumor cells to kill them. During the period of the 1950s to 1970s, viral oncolytic effects were demonstrated through studies using adenoviruses and mumps viruses, but eventually failed to draw attention for decades due to its transient efficacy and toxic side effects. However, there was a significant advance in research on an oncolytic virus in the 1990s, since the development of some viruses having improved safety than previous viruses such as thymidine kinase (TK)-deficient mutant of Herpes simplex virus-1 (HSV-1) and an adenovirus mutant, dl1520 (ONYX-015) replicating selectively in p53-defective tumor cells (Jagus R, et al., Int. J. Biochem. Cell. Biol., (1999) 31:123-138; Bischoff J R, et al., Science, (1996) 274(5286):373-376). Until now, about 80 oncolytic viruses have been developed and expected to be promising tools for anticancer therapy.
There have been several approaches in order to enhance the selective replication of viruses in tumor cells and to maximize their oncolytic effects.
The first one is to use a wild-type virus such as Newcastle Disease Virus (NDV) or reovirus which can replicate in tumor cells intactly. These viruses are generally called RNA viruses since they selectively replicate in tumor cells where RNA-activated protein kinase R (PKR)/interferon is deficient.
The second is to enhance the affinity between viruses and tumor cells by bio-engineering. Recently, protein and genetic engineering allow to add a high affinitive ligand, receptor, or antibody to viruses and to give a capsid protein such as fiber high tropism for tumor cells by adding a tumor cell-affinitive ligand thereto.
The third is to introduce modification or deletion into a viral gene to inhibit viral replication and toxicity in normal cells. E1A, one of the adenovirus primary proteins, usually binds to retinoblastoma protein (pRb) in infected cells. The failure of the binding between pRb and E1A by genetic modification results in releasing pRb. In normal cells, the free pRb preferentially binds to E2F-1 which leads to decrease E2F-1 transcriptional activity on viral replication. However, in tumor cells, since pRb gene usually has the mutation and does not bind to E2F regardless of E1A modifications, E2F freely activates transcription for viral replication and cytolysis in spite of viral infection. Another mutant adenovirus, E1B55K gene-deficient adenovirus, is also known to have reduced replication and toxicity in normal cells since it does not have E1B55K gene which inhibit the function of p53 as a tumor suppressor.
The fourth is to edit a viral vector by insertion of foreign genes or deletion of viral genes for enhancing the oncolytic effect. Adenoviral gene E1B19K is known to share homology with Bcl-2 inhibiting cell death, and it was reported that deletion of E1B19K promotes apoptosis of infected cells and enhances the antitumor activity. Further, there has also been an approach for maximizing the antitumor activity by introducing the genes encoding a cytolytic or anti-angiogenic factor such as G-CSF or IL-12 into adenovirus vector.
Finally, the fifth is to utilize a tumor-specific promoter which enables a gene essential for viral replication, e.g., E1A, to be expressed only in tumor cells. Until now, there have been developed several tumor-specific promoters derived from tumor marker genes such as carcinoembryonic antigen (CEA), α-fetoprotein, prostate-specific antigen (PSA), and telomerase (TERT).
However, according to recent studies, it has been reported that the mechanisms of tumorigenesis and metastasis are very diverse and the moiety of tumor cells frequently deviates from these mechanisms. In light of the fact that it is difficult to restrict oncolytic viruses into tumor cells and to increase the oncolytic effect by a single above-mentioned method, it is needed to maximize the therapeutic effect by combining at least one of the methods mentioned above.
Wnt is one of the representative oncogenic factors, which is a vertebrate homologue to the Drosophila segment polarity gene, wingless. Wnt 1 (wingless/int-1) was first identified in mouse mammary tumors induced by MMTV (mouse mammary tumor virus). Likewise, the increased expressions of Wnt2 and Wnt5 were reported in prostate, colon and mammary tumors. In particular, abnormal regulation of Wnt expression in colorectal tumorigenesis is well investigated. Constitutive activation of Wnt has been found in about more than 90% of colorectal cancer which gives rise to the accumulation of β-catenin in nucleus, and the nuclear β-catenin binds to transcription factor TCF to activate the transcription of Wnt target genes. Examples of the Wnt target genes include cell proliferation-involved c-Myc and Cyclin D1, anti-apoptotic factors COX-2 and PPARδ, tumor cell invasion-involved MMPs, growth factors c-met, VEGF and BMP-4. The constitutive activation of Wnt signaling pathway in colorectal cancer is mostly induced by phosphorylation of β-catenin, or the mutation of APC, Axin, or GSK3β, each of which plays an important part in the ubiquitin-related degradation of β-catenin. Recent evidences also suggest that β-catenin participates in mRNA splicing and stabilization by direct binding to pre-mRNA.
Among transcription factor E2F family, E2F-1 was first identified as an E1A-transactivation factor which binds to adenoviral E2A. E2F target genes contain the E2F binding motif of TTT(C/G)CGCG in their promoter regions, and can simultaneously regulate two contradictory phenomena, i.e. cell proliferation and apoptosis. Among currently known E2F target genes, genes encoding cell cycle regulatory factors such as Cyclin E, Cyclin A, Cyclin D, cdc2, and cdc25A; and genes encoding enzymes involved in DNA synthesis such as DHFR (dihydrofolate reductase), DNA polymerase α, and thymidine kinase (TK) are known to be associated with cell proliferation, while genes such as Apaf1 (apoptosis protease-activating factor 1), p′73, and ARF are known to be associated with apoptosis. E2F family binds to pRb called as pocket protein or pRb-related proteins, p107 and p130. Among six members of E2F family currently known, E2F-1, E2F-2, and E2F-3 expressed specifically in the G1/S phase bind with pRb to act as transcriptional activators. In contrast, E2F-4 and E2F-5 bind with p107 and p130 to inhibit the transactivation of E2F target genes. Finally, E2F-6, which lacks both the transactivation domain and the pocket protein-binding domain unlike other E2Fs, is known to inhibit the transactivation of E2F target genes. E2F-1 is generally activated for the transcription of target genes by release from the binding with pRb in case that: 1) pRb is phosphorylated by Cyclin D4/Cdk4 which is activated in G1/S phase; 2) E1A expressed in infected cells with adenovirus competitively binds to pRb; and 3) E2F binding site of pRb is mutated in tumor cells, etc. The activated E2F-1 may facilitate cell proliferation by promoting G1/S transition phase, or induce apoptosis by the transactivation of ARF to inhibit p53 suppressor MDM2.
As described above, the genetic mutation in WNT/β-catenin or malfunction of the E2F/pRb signal transduction is found so frequently in tumor cells, which causes the nuclear accumulation of β-catenin-TCF complex or E2F. Further, the cross-linking between WNT/β-catenin and E2F/pRb signal pathway has been examined via Axin2, Siah1, etc., which may be taken as an excellent tumor marker.
DNMT1 (DNA-methyltransferase 1) is an enzyme that adds methyl groups to DNA, which involves in the methylation of the CpG sites in the transcriptional regulatory region along with HDAC (histone deacetylase). The hypermethylated gene binds with a histone protein to form a tight complex of DNA and protein (heterochromatin), resulting in suppression of DNA transcription.
The present inventors have endeavored to identify a novel tumor-specific promoter in the upstream region of DNMT1 and to construct an adenovirus vector comprising the promoter, which has led the finding that the recombinant adenovirus produced from the vector has a high oncolytic activity.